Use of strains of Streptococcus thermophilus which are incapable of hydrolyzing urea in dairy products

ABSTRACT

The invention relates to the use of at least one strain of  Streptococcus thermophilus  which is incapable of hydrolyzing urea in the manufacture of cheese or fermented dairy products such as yogurts in order to obtain an acidification kinetic which is independent from the content of various components of the milk.

The present invention relates to controlling the acidification kineticof milk during the manufacture of cheeses or fermented milks such asyoghurts, through the use of Streptococcus thermophilus bacteria whichare at least partially, preferably totally, incapable of hydrolyzingurea.

Streptococcus thermophilus is a thermophilic lactic bacterium used as alactic ferment in the dairy industry. Used first of all for themanufacture of fermented milks such as yoghurt, it is now increasinglyused in cheese production.

This bacterium converts lactose into lactic acid, and through this hasan acidifying activity. In the case of cheeses notably, thisacidification not only encourages the action of the rennet and thesynaeresis of the curds but also inhibits the growth of many undesirablebacteria, certain of which are pathogenic bacteria, and even allowstheir elimination at a greater or lesser speed.

The acidifying activity of this bacterium is however accompanied by aurea hydrolysis activity, an activity that affects the acidificationkinetic. Tinson et al (1982a) showed that the urea hydrolysis reaction,giving-carbon dioxide and ammonia, resulted in a temporary decrease inthe acidification speed, measured by means of a pH probe. The authors ofthis article conclude therefrom that the changes in pH cannot be used tomeasure the lactic acid production in S. thermophilus cultures, sincethe results that would be obtained would be erroneous owing to theproduction of ammonia. Furthermore, Spinnier and Corrieu in 1989observed that the addition of urea led to a drop in the acidificationspeed.

On an industrial scale, the hydrolysis of urea by Streptococcusthermophilus poses a number of problems.

This is because, in cheese manufacturing for example, the technologicaloperations (cutting of the curds, stirring, etc.) must take place atgiven values of pH, but in practice these operations are generallycarried out at predetermined times. Therefore the variations inacidifying activity due to urea hydrolysis lead to defects andsignificant variability in the cheeses (texture, moisture level,ripening). Martin et al (1997) thus observed that the variations in ureacontent caused changes in the acidification kinetics and in the textureof Reblochon type cheeses, confirming the results obtained by Spinnierand Corrieu (1989).

Moreover, the production of ammonia increases the time necessary toreach a given pH. This results in the equipment being tied up for longerand in an increase in the risk of contamination by undesirablemicro-organisms.

Furthermore, it is desirable that the cheese-making whey does notcontain an excessive amount of ammonia, since this whey is often used inanimal feed.

This phenomenon is difficult to control, notably since the urea contentof milk is variable (generally from 2 to 8 mM) and depends in particularon the feeding of the livestock. To overcome this problem, Martin et al(1997) proposed measuring the urea content of the milk and then adaptingthe manufacturing parameters. However, the use of such a ureaquantitative analysis system would be highly constraining, and would notin any case resolve the drawbacks due to slowing down of theacidification speed in the presence of urea (equipment tied up for alonger time, increase in the risks of contamination, etc.) and to a highammonia content of the whey.

The authors of the present invention have revealed that the use ofStreptococcus thermophilus strains not, or not totally, hydrolyzingurea, as lactic ferments in the production of dairy products, made itpossible to solve the aforementioned problems. These strains aredesignated “ur(−) strains” in the remainder of this application.

Until now, the only ur(−) Streptococcus thermophilus strains describedare the CNRZ 407 strain (Juilliard et al, 1988) and the mutant strainisolated by Tinson et al (1982b). However, the information knownrelating to these two strains does not allow the technologicalimportance of ur(−) strains to be realized.

One object of the present invention is therefore the use of at least onestrain of Streptococcus thermophilus which is at least partially,preferably totally, incapable of hydrolyzing urea, during themanufacture of cheeses or fermented dairy products such as yoghurts, inorder to obtain an acidification kinetic which is substantiallyindependent of the content of the milk in terms of its constituents.

Within the context of the present invention, “the acidification kinetic”means the variation in pH of the fermentation medium as a function oftime.

“Content of the milk in terms of its constituents” means in particularthe urea content of the milk, which differs from one milk to another,depending on the origin of the animal or its feed. It also means thecontent of the milk in terms of other constituents which are involved inthe metabolism of urea. Amongst these constituents can be cited forexample nickel or cobalt. These constituents may be present naturally inthe raw material used (the milk) or may have been added.

Another object or the invention is a method for obtaining, during themanufacture of cheeses or fermented dairy products such as yoghurts, anacidification kinetic which is substantially independent of the contentof the milk in terms of its constituents, in which there is incorporatedwith the milk at least one strain of Streptococcus thermophilus which isat least partially, preferably totally, incapable of hydrolyzing urea.

The ur (−) Streptococcus thermophilus strains used in accordance withthe present invention can be obtained by a mutagenic treatment or byspontaneous mutation, or also be isolated in nature.

The strains 298-K and 298-10, which are respectively a spontaneousmutant and a mutant obtained after mutagenic treatment, were registeredat the CNCM on 14 Sep. 1999 under the numbers I-2311 and I-2312,respectively.

Any ur(−) strain cultured according to the protocol of Tinson et al(1982b), or preferably according to the protocol described in Example I,can also be used.

The ur (−) Streptococcus thermophilus strains can be used alone or in amixture with other micro-organisms such as lactococci, lactobacilli, orany other micro-organism usable in the dairy industry.

The authors of the present invention have shown that the importance ofthe ur(−) Streptococcus thermophilus strains is multifaceted. In fact,they have revealed that the ur(−) mutants make it possible not only tohave control over the variations in acidification kinetics, but thatthey are moreover stable and exhibit good growth in milk.

Furthermore, the ur(−) strains make it possible to obtain acidificationkinetics of milk which are regular, do not exhibit any temporary slowingdown, and are a function of the area concentration, unlike the kineticsobserved with the ur(+) strains.

The ur(−) strains do not produce ammonia during their growth in milk,which is advantageous from the point of view of using the whey in animalfeed.

Finally, the strains selected for their ur(−) phenotype surprisinglyhave variable acidifying characteristics, compared with theacidification kinetics observed with the parent strains.

“Variable acidification kinetic” means an acidification kinetic which isfor example faster or slower compared with the acidification kineticsobserved with the parent strains. “Heterogeneity” between theacidification kinetics of the different ur(−) mutants with regard to theparent strains can also be spoken of.

Another object of the invention is therefore a method of selectingStreptococcus thermophilus strains useful during the manufacture ofcheeses or fermented dairy products, in which mutant strains ofStreptococcus thermophilus which are at least partially, preferablytotally, incapable of hydrolyzing urea, allowing an acidificationkinetic to be obtained which is substantially independent of the contentof the milk in terms of its constituents, are selected for their abilityto acidify a milk according to acidification kinetics which are variablecompared with the acidification kinetics of the parent strains.

In general terms, the choice of the acidifying properties of the ur(−)strains can be made as a function of the cheese or fermented milkmanufacturing technology for which these strains are used.

Thus, certain ur(−) strains are characterised more particularly by anabsence of the post-acidification phenomenon.

For other strains, the time necessary to reach a given pH proves to beshorter than for the parent ur(+) strains. Thus, this property makes itpossible to seed the milk with a ur(−) mutant strain at a rate lowerthan the rate generally used for the parent ur(+) strain. This rate canbe around 25%, perhaps even around 50% lower compared with the rate thatwould be used for the parent strain.

One object of the present invention is therefore a method according tothe invention, in which there is incorporated with the milk at least onemutant strain of Streptococcus thermophilus which is at least partially,preferably totally, incapable of hydrolyzing urea, at a seeding ratelower than the seeding rate used for the parent strain of Streptococcusthermophilus capable of hydrolyzing urea.

The figures and examples below illustrate the invention without limitingthe scope thereof.

LEGEND FOR THE FIGURES

FIGS. 1A–1B depicts acidification curves for reconstituted skimmed milk,obtained with the ur(+) strain RD298 and with the spontaneous ur(−)mutants (FIG. 1A) or those obtained after treatment with NTG (FIG. 1B).

FIGS. 2A–2B depicts the acidification curves for reconstituted skimmedmilk, obtained with the strain ST888 and with the spontaneous ur(−)mutants (FIG. 2A) or those obtained after treatment with NTG (FIG. 2B).

FIGS. 3A–3B depicts the acidification curves for UHT skimmed milk,obtained with the strain RD298 and with the spontaneous ur(−) mutants(FIG. 3A) or those obtained after treatment with NTG (FIG. 3B)

FIGS. 4A–4B depicts acidification curves for UHT skimmed milk, obtainedwith the strain ST888 and with the spontaneous ur(−) mutants (FIG. 4A)or those obtained after treatment with NTG (FIG. 4B).

FIGS. 5A–5C depicts the acidification curves obtained with the strainRD298 (FIG. 5A) and the ur(−) mutants RD298-K (FIG. 5B) and RD298-10(FIG. 5C), on UHT skimmed milk supplemented with different amounts ofurea.

FIGS. 6A–6C depicts the acidification curves obtained with the strainRD298 (FIG. 6A) and the ur(−) mutants RD298-K (FIG. 6B) and RD298-10(FIG. 6C), on UHT skimmed milk supplemented or not with nickel (10 μg/lof NiSO₄.7H₂O).

FIG. 7 depicts the acidification curves obtained with the strain RD672and ur(−) mutants originating from this strain, on reconstituted skimmedmilk.

EXAMPLES Example 1

Method of Culturing Ur(−) Bacteria on Petri Dishes.

An agar-based medium whose composition is shown in Table 1 is preparedand poured into Petri dishes of diameter equal to 9 cm.

TABLE 1 Composition of the culture medium. Tryptone^(a) 2.5 g Pepsicmeat peptone^(a) 2.5 g Papainic soya peptone^(a) 5 g Autolytic yeastextract^(b) 2.5 g Meat extract^(a) 5 g Sugar (glucose, lactose orsaccharose) 5 g Sodium glycerophosphate.6H₂O 19 g Magnesium sulphate0.25 g Ascorbic acid 0.5 g Agar 15 g Distilled, water 1 litre ^(a)Blokarcompany ^(b)Fischer Scientific company

If need be, a cofactor of urease can be added to this medium. Adjust thepH to 7.0 and autoclave for 15 minutes at 115° C.

The St. thermophilus cells to be analyzed are seeded on this medium soas to obtain around 100 colonies per Petri dish. The cultures take placeunder anaerobic conditions at a temperature of 35–45° C., preferably37–42° C.

After two days of culture, there is poured over each Petri dish around20 ml of an agar-based solution prepared as follows: dissolve by heating15 g of agar in 1 liter of a potassium phosphate buffer solution at 50mM (pH 6) supplemented with 100 mg/l of bromothymol blue, cool thesolution to 50° C., add 10 g of urea and acidify the medium withhydrochloric acid until a yellowish-orange colour is obtained.

After solidification of the agar, the Petri dishes are incubated for 1hour at 37° C. The ur(+) clones form blue-coloured halos owing to theproduction of ammonia, whereas the ur (−) clones form yellow colonies.When the ur(−) mutants are sought, the clones not forming a blue haloare recovered and tested again on the same culture medium in order toconfirm the ur(−) characteristic. It should also be verified that thesemutants do not consume urea (or consume it only partially) when they arecultured in milk.

Example 2

Selection of Mutants for the Metabolism of Urea.

Mutants not consuming urea, or consuming it slightly, were sought fromthe RD298, RD672 and STS888 strains of St. thermophilus. Two approacheswere used. In the first approach, the mutants were sought aftertreatment with a mutagenic agent, while in the second approach,spontaneous mutants were sought.

a) Selection by Means of a Mutagenic Agent

The mutagenic treatment is carried out as described below.

The strains are cultured at 42° C. in 5 ml of M17 culture medium(Terzaghi and Sandine, 1975). The culture is stopped at the end of theexponential phase, and the cells are recovered by centrifuging and thenwashed with 100 mM (pH 7) phosphate buffer. The cells are next recoveredin 1 ml of buffer containing a variable content ofN-methyl-N′-nitro-N-nitrosoguanidine (NTG) and incubated for 1 hour at42° C. The cells are next washed twice with 5 ml of buffer and seeded onthe culture medium so as to obtain around 100 colonies per Petri dish.The culture is carried out as described previously (Example 1). Table 2describes the results obtained during 3 mutageneses.

TABLE 2 Selection of ur(-) mutants after treatment with a mutagenicagent (NTG). Viability (% of NTG cells Number Number St. concen- havingof of ur(⁻) Proportion thermophilus tration survived colonies clone ofur(⁻) strain used (μg/ml) the NTG) cultured obtained clones (%) ST888 2010 980 11 1.1 ST888 5 48 1000 5 0.5 RD672 50 41 10600 41 0.4 RD298 50 163200 15 0.5b) Selection of Spontaneous Mutants

In a population of micro-organisms, there often exist spontaneousmutants for a gene or a given characteristic. This type of mutant is ofgreat interest, since the fact that no mutagenic agent has been usedeliminates the risk of causing non-sought-after mutations (mutationsother than for the characteristic studied), which might impair thetechnological abilities of the strains. However, the frequency ofspontaneous mutants within a population for a given characteristic isgenerally very low, of the order of 1 in 1 million (variable dependingon the strain and characteristic). Therefore the selection ofspontaneous mutants generally requires either the development of amethod making it possible to culture a very high number of clones, orthe definition of a procedure for enriching mutants. No procedure forenriching ur(−) mutants has a priori been described. Moreover, giventhat the procedure of culturing on Petri dishes does not allow theanalysis of more than 100 colonies of St. thermophilus per dish, theselection of spontaneous mutants might have been expected to beunfeasible, since it would have been necessary to culture severalthousand, perhaps even tens of thousands, of Petri dishes, in order tohave chances of isolating a spontaneous mutant. However, the authors ofthe present invention noticed that, in the St. thermophilus cultures,the proportion of spontaneous ur(−) mutants was high (around 1 in 2500for ST888, 1 in 4000 for RD672 and 1 in 1200 for RD298), and that it istherefore possible to easily isolate this type of mutant (Table 3).

TABLE 3 Selection of spontaneous ur(−) mutants. The protocol used is thesame as that described in paragraph a) “selection by means of amutagenic agent”, except that the mutagenic agent is omitted. St. Numberof Number of Proportion of thermophilus colonies ur(−) clones ur(−)clones strain cultured obtained (%) ST888 16000 6 0.04 RD298 7400 6 0.08RD672 24000 6 0.03

47 of the 90 mutants obtained were studied. The results relating tostability, enzymatic characterization and the acidifying behaviour ofthese mutants are described below.

Example 3

Properties of the Ur(−) Mutants.

a) Stability of the Mutants

In order to be able to be usable in an industrial context, the ur(−)mutants must be stable. However, no data existed as regards thestability of ur(−) mutants of St. thermophilus. The authors of thepresent invention studied the stability of 47 mutants originating fromthe strains ST888, RD672 and RD298. The strains were subcultured dailyin 10 ml of M17 culture medium, for 20 days. The cultures wereinoculated at 1% and incubated at 42° C. The set of 20 subculturesrepresents around 130 generations. After the 20^(th) subculture, thestrains were seeded in milk and it was determined whether or not theyconsumed urea (cultures of 15 hours at 42° C.). The results are shown inTable 4. It should be noted that the ur(−) mutants, whether they areobtained by a mutagenic treatment or are spontaneous mutants, are highlystable. In fact, only two reversions were detected for the 47 mutantstested.

TABLE 4 Study of the stability of the ur(−) mutants. The ureaconsumption was tested during cultures on milk, after 20 successivesubcultures in M17 culture medium. St. Number of Number of mutantsthermophilus ur(−) mutants consuming urea after strain Mutation tested20 subcultures ST888 NTG 6 1 ST888 Spontaneous 6 0 RD298 NTG 5 0 RD298Spontaneous 6 0 RD672 NTG 19 0 RD672 Spontaneous 5 1 Total / 47 2b) Enzymatic Characterisation of the Mutants

The strains studied were cultured for 24 hours, under anaerobicconditions and at 37° C., in a liquid culture medium whose compositionis shown in Table 5. The cells were recovered by centrifuging, washed inbuffer (HEPES 50 mM-EDTA 1 mM, pH 7.5), and then recovered in a volumeof buffer representing 2% of the volume of the culture. The ureasicactivity was then measured on acellular extracts (treatment of the cellsin a ball mill and recovery of the supernatant from centrifuging for 5minutes at 20,000 g).

TABLE 5 Composition of the culture medium used for preparing theextracts. Tryptone^(a) 10 g Autolytic yeast extract^(b) 5 g Sodiumglycerophosphate.6H₂O 19 g Ascorbic acid 500 mg Magnesium sulphate 250mg Nickel sulphate.7H₂O 10 mg Glucose 10 g Distilled water 1 litre^(a)Blokar company ^(b)Fischer Scientific company

Adjust the pH to 7.0 and autoclave for 15 minutes at 115° C.

The ureasic activity measurements were carried out at 37° C., in HEPES50 mM—EDTA 1 mM (pH 7.5) buffer. The reaction is triggered by theaddition of 25 mM of urea, and the ammonia produced in 20 minutes isanalyzed quantitatively, using Nessler's reagent. The results areexpressed in units (U) of urease activity (one unit corresponds to onemicromole of ammonia produced per minute) per milligram of protein.

Table 6 shows the activity values obtained. The ur(−) mutants did notexhibit any detectable ureasic activity, with the exception of themutants 298-3.17 and 888-1.5. These correspond to mutants having a ur(+)phenotype in the presence of nickel and a ur(−) phenotype in the absenceof this compound. Now, the culture medium used for preparing theacellular extracts contained nickel sulphate. In these two strains, themutation probably focuses on the nickel transport system or the systemallowing its incorporation into the active site of the urease.

These strains of St. thermophilus could also exhibit a ur(−) phenotypeon account of an inability to transport urea. Such strains wouldtherefore always possess a measurable ureasic activity in acellularextracts.

TABLE 6 Measurement of the ureasic activity of acellular extractsobtained from the parent strains and from the ur(−) mutants. ParentUreasic Parent Ureasic Parent Ureasic strain activity strain activitystrain activity Mutant (U/mg) Mutant (U/mg) Mutant (U/mg) RD298 0.94RD672 1.08 ST888 0.95 298-10 N.D. 672-18(0) N.D. 888-A N.D. 298-K N.D.672-47(0) N.D. 888-B N.D. 298-I N.D. 672-54(0) N.D. 888-C N.D. 298-JN.D. 672-19(0) N.D. 888-D N.D. 298-L N.D. 672-31(0) N.D. 888-1 N.D.298-M N.D. 672-59(50) N.D. 888-2 N.D. 298-N N.D. 672-62(50) N.D. 888-2.6N.D. 298-3.9 N.D. 672-61(50) N.D. 888-2.11 N.D. 298-3.3 N.D. 672-33(50)N.D. 888-2.9 N.D. 298-3.16 N.D. 672-55(50) N.D. 888-1.13 N.D. 298-3.170.58 672-53(50) N.D. 888-1.8 N.D. 672-70(50) N.D. 888-1.5 0.42672-20(50) N.D. 672-50(50) N.D. 672-34(50) N.D. 672-22(50) N.D.672-24(50) N.D. 672-10(50) N.D. 672-36(50) N.D. 672-60(50) N.D.672-21(50) N.D.c) Acidifying Behaviour of the Mutants

In order to demonstrate the technological importance of the ur(−)strains, the authors of the invention compared their acidifyingcharacteristics with those of the corresponding parent strains.

The following results were observed:

-   -   unlike the parent strains, the ur(−) mutants do not exhibit a        temporary slowing down of the acidification speed due to        hydrolysis of the urea; their acidification curves are therefore        more regular;    -   the kinetics of acidification of the milk by the ur(−) mutants        are little affected or not affected by the urea, nickel and        cobalt contents;    -   furthermore, a high variability of the acidifying activities        between the ur(−) mutants is observed, compared with the        acidifying activities of the parent strains.

A breakdown of the results obtained is shown below. The cultures wereseeded at 1% with a preculture carried out on sterilized reconstitutedskimmed milk, then cultured at 37° C.

-   -   Cultures in reconstituted skimmed milk:

The milk was reconstituted at 100 g/l and pasteurized for 10 minutes at90° C.

After around 2 hours of culture, a rise in pH in the culture of thestrain RD298 is observed (FIG. 1). The 6 spontaneous mutants have a veryregular acidification curve, with no pH rise nor temporary slowing downof the acidification speed. At certain times in the culture, the shiftin acidification compared with the parent strain reaches almost 4 hours.This therefore allows a given value of pH to be reached more quickly.The importance of this observation is major: if it is wished to reach agiven pH without reducing the incubation time, a ur(−) strain can beused, reducing the amount of seeding compared with the amount used witha ur(+) strain. Certain of the mutants obtained after treatment with NTGhave a behaviour similar to the spontaneous mutants; others acidity themedium more slowly (298-3.3) or more quickly (298-10).

With the exception of the mutant 888-1 the ur(−) spontaneous mutants ofST888 have the same acidification curve. As for RD298, a more regularand faster acidification is observed with the mutants (FIG. 2).

-   -   Cultures in UHT sterilized skimmed milk (Lactel®):

As for the cultures carried out in reconstituted milk, a temporary haltin the lowering of the pH is observed with the strain RD298, thisphenomenon being absent in the cultures of the spontaneous ur(−) mutants(FIG. 3).

The ur(−) mutants isolated from ST888, whether they are spontaneous orobtained by treatment with NTG, have an acidification curve more regularthan that of the parent strain (FIG. 4).

-   -   Effect of variations in the composition of the milk on the        acidification curves:

The strain RD298, and the ur(−) mutants 298-K and 298-10, were culturedon UHT sterilized skimmed milk supplemented or nor with differentamounts of urea. The initial urea concentration of the milk was equal to3 mM and the urea contents of the different cultures were containedwithin the variation zones that are usually observed with cow's milk. Itshould be noted that, unlike the ur(−) mutants, the acidification curvesobtained with the parent strain are highly dependent on the urea contentof the milk (FIG. 5).

The authors of the present invention also observed that theacidification curves obtained with the parent strain are dependent onthe nickel and cobalt content of the milk, which is not the case for theur(−) mutants (FIG. 6).

-   -   Ammonia production:

In all the cultures described previously, it was observed that thestrains RD298 and ST888 produced ammonia and hydrolyzed all the ureacontained in the milk. No ammonia production was observed with themutants. This indicates that urea is the main substrate used by St.thermophilus for producing ammonia. Thus the use of ur(−) strains makesit possible to avoid any ammonia production due to St. thermophilusduring cheese manufacture. Consequently, the ammonia contents ofcheese-making wheys can be limited.

-   -   Variability of the acidifying activities:

The authors of the present invention observed interestingly that thecurves of acidification in reconstituted skimmed milk obtained with anumber of ur(−) mutant strains had large variations compared with thecurve obtained with their parent strain.

FIG. 7 thus shows the acidification curves for reconstituted skimmedmilk, obtained with the strain RD672, and with ur(−) mutants originatingfrom this strain.

The strain RD672 is not very acidifying (solubilized soft cheese typetechnology). The mutant 672-47(0) is distinctly more acidifying than theparent strain, while the mutant 672-36(50) has a fairly similaracidification kinetic. The mutant 672-70 (0) is distinctly lessacidifying than the parent strain and the mutant 672-24(50) is a littleless acidifying than the parent strain.

Example 4

Manufacture of “Solubilized Soft Cheese” Type Cheeses Using Either theUr(+) Industrial Strain Rd298 or the Ur(−) Mutant Strain 298-10 (amutant of RD298).

a) General Points

Under the generic name cheese, there is found a very large number ofproducts, having a technology, a flora and organoleptic properties whichare very diverse.

Technologically speaking, cheese results in the first place from thecoagulation of milk obtained by renneting, which will be followed bydraining of the coagulum thus obtained (mechanical operations such ascutting, stirring and turning). During manufacture, the growth of theadded ferments will cause a lowering of the pH of the coagulum. Theacidification kinetic (the change in pH as a function of time) and thedrainage kinetic condition the final composition of the curds andtherefore the intrinsic characteristics of the cheeses. This is why, fora given technology, having control over the acidification and drainagekinetics is essential.

b) Specific Features of the “Solubilized Soft Cheese” Technology Used.

The manufacture of “solubilized soft cheese” type cheeses corresponds tothe use of a technology with enzymatic dominance (important function ofthe rennet) with specific manufacturing temperature profiles, such asthat described in Table 7.

The conduct of the draining is characterised by:

-   -   considerable acidification at the start of the method which        conditions the draining level. The acidification is provided by        Streptococcus thermophilus: the target pHs to be reached at the        different manufacturing stages are summarized in Table 7;    -   fast removal of the whey increased by mechanical operations        (cutting, stirring and moulding of the coagulum);    -   operations facilitating the removal of the whey (turning).        c) Monitoring of the Cheese Manufacturing

Table 7 summarises the different technological steps of themanufacturing carried out and shows the process times which werenecessary in each test to reach the target pHs of each of these steps.

Two distinct milks were used, one containing less than 1 mM of urea andthe other 5 mM of urea. The ferments used consisted either of theindustrial strain RD298 known for its ability to hydrolyze urea, ur(+),or the strain 298-10, a spontaneous mutant of this strain lacking thisurea hydrolysis ability, ur(−).

Monitoring the acidification of the milk containing a very small amountof urea (less than 1 mM) shows that the two strains used allow thetarget pHs of each step to be reached in approximately identical times.Similarly, these objectives are achieved with the ur(−) strain 298-10when the manufacturing milk contains significant amounts of urea (5 mM).On the contrary, in order to meet the target manufacturing pHs with thestrain RD298 in the milk containing 5 mm of urea, the process times havehad to be considerably lengthened.

This study therefore demonstrates the certain technological advantage ofthe ur(−) mutant 298-10 compared with the ur(+) industrial mother strainRD298.

TABLE 7 Technological characteristics of a “solubilised soft cheese”type cheese manufacture and technological description of manufacturingcarried out with the ur(+) strain RD298 or the ur(−) strain 298-10 usedas ferments from milk containing either 5 mM of urea or less than 1 mMof urea. Process Actual process time (min) Manufacturing time Milk withless than Milk containing 5 Manufacturing temperature Target pHobjectives 1 mM of urea mM of urea stage (° C.) (±0.05) (±10 min) RD298298-10 RD298 298-10 Milk 38 ± 0.5 6.48  0 ± 10 0 0 0 0 Renneting 6.40 70 ± 10 70 60 100 60 Moulding 6.30 120 ± 10 120 110 140 110 1^(st)turning 35 ± 0.5 6.20 180 ± 10 190 170 280 170 2^(nd) turning 26 ± 0.55.50 300 ± 10 310 310 450 310 3^(rd) turning 20 ± 0.5 5.25 540 ± 10 540530 700 530

BIBLIOGRAPHY

-   Juillard V., Desmazeaud M. J., Spinnier H. E. 1988. Revelation of a    ureasic activity in Streptococcus thermophilus. Canadian Journal of    Microbiology. 34: 818–822.-   Martin B., Coulon J. B., Chamba J. F., Bugaud C. 1997. Effect of    milk urea content on characteristics of matured Reblochon cheeses.    Lait. 77: 505–514.-   Spinnier H. E., Corrieu G. 1989. Automatic method to quantify    starter activity based on pH measurement. Journal of Dairy Research.    56: 755–764.-   Terzaghi B. E., Sandine W. E. 1975. Improved medium for lactic    streptococci and their bacteriophages. Applied Microbiology. 29:    807–813.-   Tinson W., Broome M. C., Hillier A. J., Jago G. R. 1982a. Metabolism    of Streptococcus thermophilus. 2. Production of CO2 and NH3 from    urea. Australian Journal of Dairy Technology. 37: 14–16.-   Tinson W., Ratcliff M. F., Hillier A. J., Jago G. R. 1982b.    Metabolism of Streptococcus thermophilus. 3. Influence on the level    of bacterial metabolites in cheddar cheese. Australian Journal of    Dairy Technology. 37: 17–21.

1. A method for obtaining, during the manufacture of a dairy productselected from the group consisting of cheeses and other fermented dairyproducts, an acidification kinetic which is substantially independent ofthe content of the milk in terms of constituents which are involved inthe metabolism of urea, said method comprising incorporating with themilk at least one strain of Streptococcus thermophilus which isincapable of hydrolyzing urea.
 2. The method according to claim 1, inwhich the acidification kinetic is substantially independent of the ureacontent of the milk.
 3. The method according to claim 1, in which theacidification kinetic of the milk is substantially independent of thenickel or cobalt content of the milk.
 4. The method according to claim1, in which the acidification kinetic of the milk does not exhibit anytemporary slowing down.
 5. A method according to claim 1, in which thereis incorporated with the milk at least one mutant strain ofStreptococcus thermophilus which is incapable of hydrolyzing urea, at aseeding rate lower than the seeding rate used for the parent strain ofStreptococcus thermophilus capable of hydrolyzing urea.
 6. A methodaccording to claim 1, in which the Streptococcus thermophilus strain isthe strain 298-K registered at the CNCM under number I-2311.
 7. Themethod according to claim 1, in which the Streptococcus thermophilusstrain is the strain 298-10 registered at the CNCM under the numberI-2312.